*3.3. Generation of Genetic Variability by Recombination between T. cruzi RHS Sequences*

In the phylogenetic analysis, we found sixty-five RHS sequences distributed in branches with low bootstrap values, which were included in the unclassified groups. Due to the high number of unclassified sequences, we investigated whether recombination events had also occurred in these sequences. We used the Circos plot to map the recombination events between RHS with a single link connecting each pair of paralogs (Figure 1). We identified 53 recombination events in 139 RHS sequences that were confirmed by at least six of the seven algorithms of the RDP4 package (Figure 4). We found that about 60% of the recombination events occurred in the unclassified sequences. Thirty-two unclassified RHS sequences were involved in the recombination events. The size of the fragment inserted into the target sequence by recombination is quite variable, and it may represent approximately 4% of the entire RHS gene. The recombination between the RHS genes results in mosaic structures that can contain up to three fragments of different RHSs inserted in the target sequence.

The recombination events occurred in different regions of RHS including the coding regions of the amino- and carboxy-terminal portions, as well as in the central region of the protein. Most recombination events were detected in the RHS sequences of group 3 that served as donors into unclassified sequences and eventually into sequences from other RHS groups. The recombination events occurred in specific regions, e.g., the amino-terminal coding region of RHS genes. As an example, the insertion of the same RHS sequence TcCLB.507841.14 of group 7 into the amino-terminal coding region of unclassified RHS sequences is shown (Figure 4, see recombination events 46 to 53).
